Page 1 / 10

Hemoglobin A1c Whole Blood

HPLC Analysis Kit User Manual

ZV-4001-0500-25

500

RT

D. No: ZV-4001-KK-25_Rev14
 Page 2 / 10

Table of Contents
1. INTENDED USE ......................................................................................................... 3
2. SUMMARY AND EXPLANATION ............................................................................ 3
3. IVD SYMBOLS ............................................................................................................ 3
4. TEST PRINCIPLE ...................................................................................................... 3
5. WARNING AND PRECAUTIONS ............................................................................ 4
6. STORAGE AND STABILITY ..................................................................................... 4
7. MATERIALS SUPPLIED ........................................................................................... 4
8. MATERIALS REQUIRED BUT NOT SUPPLIED................................................... 5
9. PROCEDURE NOTES............................................................................................... 5
10. PRE-SET UP INSTRUCTIONS ................................................................................ 6
11. LIMITATIONS OF THE PROCEDURE ................................................................... 6
12. SAMPLE PRETREATMENT ..................................................................................... 6
13. QUALITY CONTROL ................................................................................................. 7
14. CALCULATION OF RESULTS ................................................................................. 7
15. INTERPRETATION OF RESULTS .......................................................................... 8
16. EXPECTED VALUES................................................................................................. 8
17. HPLC-UV PARAMETERS ......................................................................................... 8
18. ANALYTICAL PERFORMANCE .............................................................................. 9
19. SAMPLE CHROMATOGRAM .................................................................................. 9
20. REFERENCES.......................................................................................................... 10

D. No: ZV-4001-KK-25_Rev14
 Page 3 / 10

1. INTENDED USE

Quantitative HPLC analysis kit for HbA1c in human whole blood samples.

2. SUMMARY AND EXPLANATION

The amount of HbA1c in whole blood is one of the most important parameters for diabetes
screening and monitoring. HbA1c is formed by combination of hemoglobin protein and sugar in
the blood. The production of HbA1c rises when the rate of blood sugar increases. Therefore, the
amount of HbA1c is directly related with high blood glucose. The different types of HbA1c
molecules can be occurred such as A1a, A1b, s-A1c, L-A1c, according to the type of sugar which
combined to the hemoglobin. The most important part of the HbA1c analysis is the best separation
of these species.

Zivak Hemoglobin A1c HPLC Analysis Kit was developed for screening and monitoring diabetes
mellitus in human blood samples. The analysis time is 1.5 minutes. And also 0.5 minutes column
washing done at analysis end. Main methods and procedures that have been selected are based
on EN ISO 13485 and 98/79/EC.

3. IVD SYMBOLS

Conformite European Manufacturer

In vitro diagnostic Order number

Sufficient for <N>

Lot number
Tests

Temperature limits

4. TEST PRINCIPLE

The whole blood specimen is diluted with hemolysis solution. Then hemolyzed samples are
injected to a cation exchange column. HbA1c is separated from other species by using a pH
gradient and ionic interactions between cation exchange group on the column material and the
hemoglobin components.

D. No: ZV-4001-KK-25_Rev14
 Page 4 / 10

5. WARNING AND PRECAUTIONS

• For in-vitro diagnostic use only.

• For professional use only.
• Read the instructions carefully, before you start.
• In case of damage of the kit package, please contact Zivak or your supplier.
• Do not use expired kits and components.
• Please check the batch no and expiry date before start.
• Protective gloves and goggles should be worn.
• Please take any necessary precautions to prevent infection with blood borne
pathogens while working with biological fluids. Appropriate bio-safety precautions and
disposal of bio-hazardous wastes should be followed.
• Please check the labels on reagent bottles. Reagents of this kit contain hazardous
material may cause eye and skin irritations.

6. STORAGE AND STABILITY

• This analysis kit can be shipped at room temperature.

• After arrival, mobile phases and reagent 1 must be stored at room temperature.
• Whole blood controls and column must be stored at 2-8 °C until reconstitution.
• All components are guaranteed until expiry date when stored at recommended
temperatures and used as described in these instructions.

7. MATERIALS SUPPLIED

Order No Volume Symbol Component

Reagent 1,
ZV-4001-05R1-25 4x2L R1
Contains aqueous buffer
Mobile Phase A,
ZV-4001-05MA-25 1 x 2.1 L MA
Contains organic solvent
Mobile Phase B,
ZV-4001-05MB-25 1 x 0.9 L MB
Contains organic solvent

ZV-4001-KK-25 1 x 1 pc KK User Guide

D. No: ZV-4001-KK-25_Rev14
 Page 5 / 10

8. MATERIALS REQUIRED BUT NOT SUPPLIED

Order No Volume Symbol Component

Calibrator Hemolysate
ZV-4001-02S1-25 1 x 3 mL Level 1 Calibrator Level 1
Calibrator Hemolysate
ZV-4001-02S2-25 1 x 3 mL Level 2 Calibrator Level 2
Control Hemolysate
ZV-4001-02K1-25 1 x 3mL
Level 1 Control Level 1
Control Hemolysate
ZV-4001-02K2-25 1 x 3 mL
Level 2 Control Level 2
Zivak Hemoglobin A1c Whole Blood HPLC Analytical
ZV-4001-02C1-25 1 x 1 pc
Column

9. PROCEDURE NOTES

Any inappropriate handling of samples or modification of the test procedure may influence the
results. The indicated pipetting volumes, incubation times, temperatures and pre-treatment steps
have to be performed strictly according to the instructions. Use calibrated pipettes and devices
only.

Once the test has been started, all steps should be completed without interruption. Make sure that
required reagents, materials and devices are prepared ready at the appropriate time.

Leave aside all reagents and specimens to reach room temperature (18-25 °C) and gently swirl
each vial of liquid reagent and sample before use. Mix reagents without foaming.

Avoid contamination of reagents, pipettes and wells/tubes. Use new disposable plastic pipette tips
for each reagent, standard or specimen. Do not interchange the vial caps. Always keep vials
closed when not been used. Do not re-use wells/tubes or reagents.

Incubation time affects results. All tubes or wells should be handled in the same order and time
sequences.

D. No: ZV-4001-KK-25_Rev14
 Page 6 / 10

10. PRE-SET UP INSTRUCTIONS

Setup the Instrument
• Before start, wash the column by 1mL/min flow of %100 mobile phase B for 10 minutes.
Then activate HbA1c method and wait for 5 minutes. After conditioning step, injections can
be started. At the end of analysis, definitely wash the column with Wash Buffer before
putting it away. Above washing instructions will be automatically performed when
this kit used with Zivak HB-100 Full Automated HbA1c/Variant HPLC Analyzer.
• Keep the column at 4 °C when not used.
• Make sure the bottle of mobile phase is closed well, otherwise components of the mobile
phase could evaporate; this alters the retention times.

Preparation of Calibrators
• Calibrators are ready to use. No sample pretreatment needed.
• Calibrators must be stored at +4 °C temperature.

Preparation of Controls
• Controls are ready to use. No sample pretreatment needed.
• Controls must be stored at +4 °C de temperature.

11. LIMITATIONS OF THE PROCEDURE

Specimen collection and storage has a significant effect on the test results. Specimen volume
must be minimum 2 ml. Whole Blood samples from earlier days must be kept refrigerated before
the analysis. Whole Blood EDTA samples can be kept for 7 days at 2-8 °C and 24 hours at room
temperature.

12. SAMPLE PRETREATMENT

Sample Pretreatment (Manual)

Take 5 L patient sample into a glass vial.

Add 1 mL of Reagent 1 and vortex for 10 seconds.

D. No: ZV-4001-KK-25_Rev14
 Page 7 / 10

Inject 20 L to the HPLC-UV system.

Prepared samples are stable for 24 hours at 2-4 °C temperature.

Sample Pretreatment (Automated)

Place whole blood EDTA samples into system tray.

Analysis will automatically start after clicking Start button on software.

Injections, calculations and reporting done automatically by instrument.

Prepared samples are stable for 24 hours at 2-4 °C temperature.

13. QUALITY CONTROL

The test results are only valid if the test has been performed by following the instructions. Moreover
the user must strictly adhere to the rules of GLP (Good Laboratory Practice) or other applicable
standards/laws. All standards and kit controls must be found within the acceptable ranges as
stated on the QC (Quality Control) Certificate. If the criteria are not met, the run is not valid and
should be repeated.

Each laboratory should use known samples as further controls. In case of any deviation, the
following technical issues should be proven:

Expiry dates of (prepared) reagents, storage conditions, pipettes, devices, incubation conditions
and washing methods. It is recommended to participate at appropriate quality assessment trials.

14. CALCULATION OF RESULTS

The obtained percentage area of the analytes is used for calculation of HbA1c results. A linear
calibration curve will created with these calculated percentage areas. Calculation formula is given
below.

D. No: ZV-4001-KK-25_Rev14
 Page 8 / 10

𝐴1𝑐 𝐴𝑟𝑒𝑎 𝑋 100

%𝐴𝑟𝑒𝑎 =
(𝐴1𝑐 𝐴𝑟𝑒𝑎 + 𝐴0 𝐴𝑟𝑒𝑎)

Sample signals above the highest control level have to be confirmed by a reference method.

15. INTERPRETATION OF RESULTS

Various societies recommend different Cut-Off values for repetition of the measurement and the
application of confirmatory assays. Depending on the application of patient samples from different
populations, it is highly recommended that each laboratory establishes its own range of
normal values and that this distribution of values is coordinated with the recommendations of the
responsible society of this geographic region.

The results themselves should not be the only reason for any therapeutic consequences.
They have to be correlated to other clinical observations and diagnostic tests.

16. EXPECTED VALUES

%4.0 – %5.6 Normal

%5.7 – %6.4 Pre-diabetes
> %6.5 Diabetes

It is recommended that each laboratory establishes its own range of normal values.

17. HPLC-UV PARAMETERS

Instrument Zivak HB-100 Full Automated HbA1c/Variant HPLC Analyzer

Column Zivak Hemoglobin A1c HPLC Column
Detector UV/VIS
Wavelength 415 nm
Injection
20 µL
Volume
Column Oven
35 °C
Temperature

D. No: ZV-4001-KK-25_Rev14
 Page 9 / 10

Time Flow Rate

%A %B
(min) (µL/min)
1 00.00 1500 98 2
2 00.31 1500 98 2
3 00.32 1500 2 98
4 00.49 1500 2 98
5 00.50 1500 98 2
6 01.30 1500 98 2

18. ANALYTICAL PERFORMANCE

Intra-assay Inter-assay
Accuracy Linearity
No Analyte Precision Precision
(%) (R2)
(%CV) (%CV)
1 HbA1c 98.32 0.479 1.19 0.9985

Analytical Specificity (Cross Reactivity): No cross-reactivity was found with the typical
substances tested.

19. SAMPLE CHROMATOGRAM

D. No: ZV-4001-KK-25_Rev14
 Page 10 / 10

20. REFERENCES

1. https://www.medicinenet.com/what_is_hemoglobin_a1c_hba1c/views.htm
2. https://www.diabetes.co.uk/what-is-hba1c.html
3. https://www.webmd.com/diabetes/guide/glycated-hemoglobin-test-hba1c
4. Florkowski C., “HbA1c as a Diagnostic Test for Diabetes Mellitus-Reviewing the Evidence”, Clin
Biochem Rev, 34(2):75-83, 2013.
5. Sandler C. N., McDonnell M. E., “The Role of Hemoglobin A1c in the Assessment of Diabetes
and Cardiovascular Risk”, Cleveland Clinic Journal of Medicine, 83 Suppl 1(5):54-510, 2016.
6. James T. M., Davis J. E., McDonald J. M., Santiago J. V., Ladenson J. H., “Comparision of
Hemoglobin A1c and Hemoglobin A1 in Diabetic Patients”, Clinical Biochemistry, Volume 14,
Issue 1, 25-27, 1981.

D. No: ZV-4001-KK-25_Rev14